Background <p>Tylvalosin (acetylisovaleryltylosin), a breakthrough semi-synthetic derivative of tylosin engineered to address pharmacokinetic limitations and bacterial resistance, exhibits dual therapeutic efficacy against porcine reproductive and PRRSV-associated pneumonia and mycoplasma infections in swine. Limited research has yet to investigate pharmacokinetic profiles of tylvalosin and its metabolites. This study, therefore, aims to pioneer a robust UPLC-MS/MS method for simultaneous quantification of tylvalosin and its key bioactive metabolites, desmycosin 3-acetate and tylosin 3-acetate, in swine plasma and tissues, and to investigate the pharmacokinetic properties of different tylvalosin formulations.</p> Results <p>A robust UPLC-MS/MS method was developed and validated, showing excellent performance with good linearity, low deviation, high accuracy, minimal carry-over and matrix effects, as well as favorable stability under various conditions. Pharmacokinetic analysis following intravenous (i.v.) or intragastric administration in swine revealed that formulation I outperformed other formulations, exhibiting the highest bioavailability (<i>F</i>, 57.32%), the longest elimination half-life (<i>t</i>_<i>1/2</i>, 7.93&#xa0;h), and a delayed time to maximum plasma concentration (<i>t</i>_<i>max</i>, 3.14&#xa0;h). Dose-normalized metabolites exposures were also significantly higher for formulation I compared to other formulations. Critically, tylvalosin and its metabolites preferentially accumulated in pulmonary and lymphatic tissues at 48&#xa0;h post-administration, indicating rapid absorption, swift metabolism, and slow elimination.</p> Conclusions <p>The findings confirm the optimized therapeutic potential of tylvalosin, particularly formulation I, supported by its favorable pharmacokinetic profile and tissue distribution. This study establishes a new standard for veterinary PK-PD research and provides a PK evidence-based formulation optimization approaches to enhance treatment efficacy.</p>